Doubly-labeled 3H, 14Ctyrosines, 1-13C-tyramine or 2-14Ctyramine, administered to the stems of intact Papaver somniferum L. plants, were found to be incorporated into the morphinan alkaloids of the ...plant with comparable efficiency. 3H/14C ratios of alkaloids from plants fed the tyrosines were consistent with an almost equal conversion of this amino acid into the tetrahydroisoquinoline (TIQ) and benzyl-derived segments. Nuclear magnetic resonance (NMR) analyses of morphine isolated after administration of 1-13Ctyramine demonstrated selective labeling of C-16 of the alkaloid, indicating the conversion of this amine primarily into the TIQ-derived moiety. Morphine and thebaine labeled by 2-14Ctyramine were degraded to phenanthridines and N,N′-dimethyl ethylamines. Of the total radioactivity in the alkaloids 97% was found to be associated with the ethylamines, a distribution consistent with the NMR data. This preferential utilization of tyramine in the biosynthesis of morphinan alkaloids can be explained by the compartmentalization of intermediates and enzymes of the pathway.
Fractionation of Papaver bracteatum Arya II Lindl. latex on Renografin step gradients revealed that 43% of the dopamine was compartmentalized along with α-mannosidase (40%) in vacuoles sedimenting in ...the 2% fraction. Twenty-two percent of the dopamine was in the supernatant, but a corresponding amount (18%) of α-mannosidase was also present suggesting vacuole breakage during isolation. By subcellular fractionation of protoplasts from cultured P. bracteatum cells, the 1,000g sedimenting organelles have been identified as the major site of accumulation of the morphinan alkaloid, thebaine (99±0.8%), and the benzophenanthridine alkaloid, sanguinarine (96±3%). Although the 1,000g pellet also contained 33±4% of the total alkaloid precursor, dopamine, and half of the total vacuolar marker enzyme, α-mannosidase, 62±10% of the amine was localized in the 100,000g supernatant. A differential distribution of the alkaloids was discovered upon resolution of the lysed protoplasts on Renografin step gradients. Over 40% of the dopamine was in the supernatant with 15% in a 2% Renografin band. The remainder was evenly distributed in denser fractions of the gradient. The 4 to 8% interface, previously found to contain the largest amount of thebaine and small amounts of sanguinarine and dopamine, has been shown to be enriched in vacuoles by electron microscopy. Using a histofluorescence method, dopamine compartmentation in vacuoles of intact cultured cells was corroborated. In summary, dopamine, sanguinarine, and thebaine occur in vacuoles of different densities. A large fraction of the total dopamine in cultured cells was found in the 100,000g supernatant along with 37% of the α-mannosidase suggesting that the amine may be sequestered in more fragile vacuoles than the alkaloids. The possibility that some dopamine may be cytosolic cannot be ruled out.
Fractionation of
Papaver bracteatum
Arya II Lindl. latex on Renografin step gradients revealed that 43% of the dopamine was compartmentalized along with α-mannosidase (40%) in vacuoles sedimenting in ...the 2% fraction. Twenty-two percent of the dopamine was in the supernatant, but a corresponding amount (18%) of α-mannosidase was also present suggesting vacuole breakage during isolation. By subcellular fractionation of protoplasts from cultured
P. bracteatum
cells, the 1,000
g
sedimenting organelles have been identified as the major site of accumulation of the morphinan alkaloid, thebaine (99±0.8%), and the benzophenanthridine alkaloid, sanguinarine (96±3%). Although the 1,000
g
pellet also contained 33±4% of the total alkaloid precursor, dopamine, and half of the total vacuolar marker enzyme, α-mannosidase, 62±10% of the amine was localized in the 100,000
g
supernatant. A differential distribution of the alkaloids was discovered upon resolution of the lysed protoplasts on Renografin step gradients. Over 40% of the dopamine was in the supernatant with 15% in a 2% Renografin band. The remainder was evenly distributed in denser fractions of the gradient. The 4 to 8% interface, previously found to contain the largest amount of thebaine and small amounts of sanguinarine and dopamine, has been shown to be enriched in vacuoles by electron microscopy. Using a histofluorescence method, dopamine compartmentation in vacuoles of intact cultured cells was corroborated. In summary, dopamine, sanguinarine, and thebaine occur in vacuoles of different densities. A large fraction of the total dopamine in cultured cells was found in the 100,000
g
supernatant along with 37% of the α-mannosidase suggesting that the amine may be sequestered in more fragile vacuoles than the alkaloids. The possibility that some dopamine may be cytosolic cannot be ruled out.
By combining several established techniques we developed a method to test the specificity of mixed oligodeoxynucleotide hybridization probes and to provide the information for the design of long ...nondegenerate, and therefore more specific probes. Mixed oligodeoxynucleotide probes derived from known peptide sequences are first used to initiate primer extension reactions with poly(A)+RNA as template in the presence of three dNTPs and one ddNTP to generate cDNA transcripts of defined lengths. Comparing the lengths of the cDNA transcripts with the possible nucleic acid sequence coding for the known oligopeptide indicates whether the oligodeoxynucleotide mix hybridizes predominantly to the RNA of interest. In a second step, the oligodeoxynucleotide mix with the highest specificity is used for indirect RNA sequence analysis. This confirms the specificity of the probe and provides information to design a long, highly specific oligodeoxynucleotide probe for the gene of interest. This simple two-step-procedure helps to circumvent the time-consuming procedures of subcloning and sequencing of cross-hybridizing fragments.
